Full length human HCN1Ih channel subunit and variants

ABSTRACT

This invention relates to a DNA sequence encoding a member of the hyperpolarized activated ion channel family (HCN) and variants thereof, and the use of said sequences in assays for the measurement of gene expression. It also relates to assays for screening of I h  activators and blockers for clinical and therapeutic use in the management of human psychiatric and neurological dysfunction in the CNS, cardiovascular dysfunction of the heart, and reproductive dysfunction and/or contraception related to I h  function in testes and spermatozoa. Further, antibodies against the expressed DNA sequences and other compounds reactive with the expressed DNA sequence are also part of the invention.

FIELD OF THE INVENTION

This invention relates to a DNA sequence encoding a member of thehyperpolarised activated ion channel family (HCN) and variants thereof,and the use of said sequences in assays for the measurement of geneexpression. It also relates to assays for screening of I_(h) activatorsand blockers for clinical and therapeutic use in the management of humanpsychiatric and neurological dysfunction in the CNS, cardiovasculardysfunction of the heart, and reproductive dysfunction and/orcontraception related to I_(h) function in testes and spermatozoa.Further, antibodies against the expressed DNA sequences and othercompounds reactive with the expressed DNA sequence are also part of theinvention

BACKGROUND OF THE INVENTION

Voltage-gated ion channels play a critical role in shaping of electricalactivity of neuronal and muscle cells, and in controlling the secretionof neurotransmitters and hormones through the gating of calcium ionentry. Large families of voltage gated sodium (Na⁺), potassium (K⁺) andcalcium (Ca²⁺) ion channels have been defined usingelectrophysiological, pharmacological and molecular techniques [1, 18];they are named according to their selective permeability for aparticular cation with reference to their voltage dependence, kineticbehaviour or molecular identity. The importance of membrane voltage andion permeability in the control of cell function ensures that modulationof ion channels will invariably have important consequences for cellsand tissues, and such modulation can often be turned to therapeuticadvantage. Major indication for ion channel modulators already includecardiac arrhythmia, hypertension, anxiety, epilepsy, pain,chemotherapy-induced nausea and diabetes as well as a range of drugs indevelopment for important new indications such as neuroprotection andpsychiatry.

A variety of bodily functions such as heart beat, sleep-wake cycles,secretion of hormones and control of behavioural state depend on theaction of pacemakers, specialised cells that are able to generaterhythmic, spontaneously firing action potentials. The archetypal organdisplaying autonomic rhythmicity is the heart.

Pacemaking in the heart is accomplished by the rhythmic discharge of thesino atrial node [8, 11, 12]. The firing rate of the sino atrial node isdetermined by the diastolic depolarisation phase of the actionpotential. During this phase the membrane potential is slowlydepolarised to the threshold triggering the next action potential. Theionic conductance underlying the cardiac pacemaker depolarisation wasidentified in the late seventies and early eighties [10] and calledI_(f) (f for ‘funny’) or I_(h) (h for hyperpolarisation activated). Asimilar current was subsequently discovered in neurones, first inphotoreceptors, [2, 3, 6] and then in various central neurons eghippocampal pyramidal cells [16] where is was called I_(q) (q for‘queer’). This current was subsequently found in a wide variety ofcentral and peripheral neurons [25].

I_(h) channels have several distinctive features. Unlike mostvoltage-gated channels, they open in response to negative-going voltagesteps to potentials within the range of the normal resting potential.They conduct both K⁺ and Na⁺ ions with a three fold greater permeabilityto K⁺, yielding a reversal potential of −30 to −40 mV underphysiological conditions. As a result, the opening of these channelsnear the resting potential (˜−60 mV) generates an inward, depolarisingcurrent that is largely carried by Na⁺ [25]. Another unusual property ofthese channels is their regulation by cyclic nucleotides [11], whichspeed up the rate of channel activation by binding to an intracellularsite on the channel. In the heart, this in an important mechanismresponsible for the acceleration of heart rate in response tosympathetic stimulation. Activation of β-adrenergic receptors leads toan activation of adenylyl cyclase with the resulting increase inintracellular cAMP directly activating the I_(h) channel. cAMP bindingleads to a shift in the activation curve towards more positive voltages.This shift results in an increased inward current at a fixed membranepotential and therefore an acceleration of the diastolic depolarisation[9]. Muscarinic stimulation slows the heart rate, in part due to adecrease in cAMP level and a resulting reduction in the I_(h) current[13, 33].

In neurons, I_(h) channels have diverse functions. They were initiallyshown to be inward rectifiers; they are active near the restingpotential and pass inward current more readily than outward current,thereby helping to control of resting potential and input resistance[25]. In photoreceptors, I_(h) channels help to damp the hyperpolarisingeffect of light; they are activated by hyperpolarisation, causing thevoltage response to light to fade during the first 100-200 ms, thusproducing sensory adaptation. In many CNS neurons, activation of I_(h)channels following post inhibitory post synaptic potentials contributesto a rebound afterdepolarisation (ADP), which can trigger an actionpotential. I_(h) is also generates or contributes to ‘pacemaker’potentials that controls the rate of rhythmic oscillations, similar toits role in the heart. I_(h) has been found to regulate the rhythmicactivity of thalamic relay neurons [23] and inferior olivary neurons [5]through interaction with a T-type calcium current. The oscillatingsingle neurons are part of neuronal networks and are involved in thegeneration and modulation of rhythmic activity of these networks. A wellstudied example are spindle waves observed in the EEG during slow wavesleep, which are generated through interactions between thalamicreticular and relay neurons [4]. Regulation of I_(h) in these cells isimportant in the sleep-wake cycle. Although less well investigated,results suggest a similar role for I_(h) in the generation ofoscillations in hippocampal neurons [21, 32] and respiratory neurons ofthe preBotzinger complex of the ventrolateral medulla [26]. I_(h)channels are also expressed in dendrites where they influence the cableproperties of the dendrite and shape the time course of the EPSP as itis propagated to the soma [22]. A recent study has extended the role ofI_(h) neurons by showing that these channels can alter neurotransmitterrelease from presynaptic terminals as Crayfish neuromuscular synapses.Presynaptic cAMP generation by via serotonin receptor activationdirectly modulates I_(h) in axons that produces an increase in synapticstrength which cannot be explained solely by depolarisation of thepresynaptic membrane [7].

The genes encoding I_(h) channels were recently cloned from both mammals[19, 28, 29] and sea urchins [15]. These genes, called HCN1-4 inmammals, are members of the voltage gated K⁺ channel family. The encodedproteins contain six transmembrane segments, including a positivelycharged S4 voltage sensor and a pore-forming P region that includes theK⁺ channel signature sequence GYG. In addition, the C-terminus containsa 120-amino acid sequence that is homologous to the cAMP and cGMPbinding domains of other proteins, and is therefore the likely site forcAMP regulation of channel opening [9,20]. All four mammalian genes areexpressed in brain, with differing expression patterns [24]. While thefirst reported cloning of I_(h) was from sea urchin spermatozoa [15],the functional significance is poorly understood at present. The channelis expressed in sperm flagellum and it was postulated that it may beinvolved in the control of flagella beating. One of the cloned isoforms,HCN4, has been detected in testes suggesting that I_(h) may also have afunction in mammalian testicular and sperm function [30].

These ion channels are the target for blocking molecules for therapeuticuse in dysfunction in the CNS, cardiovascular dysfunction of the heart,and reproductive dysfunction and/or contraception related to I_(h)function in testes and spermatozoa. For instance, compounds have alreadybeen developed with the therapeutically interesting property of inducingbradycardia with minimal inotropic side effects [14, 17]. Unfortunately,there are no compounds yet described that distinguish cardiac fromneuronal isoforms, and volunteers experienced optical hallucinationsprobably due to reduced functionality of I_(h) in photoreceptors. It isclear the ability to develop agents that are selective for the CNS vsheart or vice versa requires the availability of the cloned subunits toscreen for compounds selective for subunits expressed in either neuronalor cardiac tissue.

BRIEF SUMMARY OF THE INVENTION

It is the object of the present invention to provide full-lengthfunctional human HCN1 channel subunits. Furthermore, variants of thefull-length functional human HCN1 subunit are also provided. Human HCN1channel subunits are preferred over subunits from other species sincethey are preferably used to select compounds that can be used to treatCNS disorders, cardiovascular dysfunction of the heart, and reproductivedysfunction and/or contraception related to I_(h) function in testes andspermatozoa in humans.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention concerns the full length cDNA sequence ofthe human HCN1 channel subunit (SEQ ID NO: 1). The expression of thissubunit is predominantly restricted to the CNS and has only beendetected in some peripheral tissues to a very limited extend. Thissubunit is therefore a candidate for screening compounds with aselective effect in the CNS with a reduced propensity for undesirableside effects such as bradycardia via activity at I_(h) in the heart. Incontrast with the prior art, the present invention provides thefull-length sequence of human HCN1 and sequence variants whereasprevious publications have only identified partial human HCN1 sequences.The major advantage of having a full-length sequence is that this allowsfunctional expression in in vitro systems (ie in transfected cell lines)for the identification of compounds increasing (opening the channel) ordecreasing (blocking the channel) ion flux through the expressedchannel. While expression of partial sequences may be possible, thesewill not give rise to channels with the full functional characteristicsof the full-length channel since they lack polypeptide domains thatcontribute to the functional characteristics of the channel. Inaddition, partial sequences may lack polypeptide domains with importanttarget sites for compounds that modulate channel activity. Functionalexpression of partial sequences, particularly those lacking the 5′ endsuch as those reported for human HCN1 [1] is additionally problematicsince they lack the sequence encoding the N-terminal signal peptiderequired for directing the nascent polypeptide to the endoplasmicreticulum where all nascent plasma membrane bound proteins aresynthesised. The inherent advantage of an assay based on channelfunction, rather than, say a binding assay, is that a functional assayallows for the screening of compounds that interact with the channel aseither blockers or openers. Binding assays give no such information, nordo they allow the identification of allosteric or use-dependentmodulators of the channel.

By the term “full-length” as used herein we mean a DNA sequence thatcontains a complete open reading frame, beginning with a start codon atits 5′ end, preferably with a Kozak consensus, downstream to an in-framestop codon.

The full-length DNA sequence for HCN1 (SEQ ID NO: 1) was obtained usinga combined molecular biology and bioinformatics approach. Initialscreening of a proprietary database with the published human HCN1sequence [1] identified a single clone according to SEQ ID NO: 9 thatmatched at the 3′ end. Further analysis of the predicted amino acidsequence of this clone (SEQ ID NO: 10) confirmed that it encoded the 3′end of human HCN1 with an in-frame stop codon. Comparison with thefull-length murine HCN1 sequence indicated that the published humansequence lacks approximately 330 bases of 5′ sequence upstream to thetranslational initiation codon. Several approaches were adopted toattempt to obtain the missing 5′ end. These included cDNA libraryscreening, genomic library screening and 5′ RACE-PCR. RACE-PCRconsistently generated a further 153 bp (SEQ ID NO: 13) upstream of thepublished human HCN1 sequence. A search of genomic databases with thisnew 5′ end sequence identified a BAC clone (RP11-398G9) in the updatedEMBL high throughput genomic (HTG) database (em62htgnew). Sequenceanalysis of this clone suggests that it contains an in-frame initiationcodon and an open reading frame homologous to murine HCN1. The 5′ end ofhuman HCN1 is extremely GC-rich (approximately 80%) thus explaining whyconventional means of cloning this sequence proved fruitless.

Thus, sequence from a combination of newly identified genomic BAC clone,RACE-PCR, published sequence and the clone obtained from the proprietarydatabase have been assembled to generate a full length cDNA encoding afull length human HCN1 Ih channel subunit (SEQ ID NO: 1).

In another aspect of the invention, the sequences of the presentinvention can be used to derive primers and probes for use in DNAamplification reactions in order to perform diagnostic procedures or toidentify further, neighbouring genes which also may contribute to theexpression of HCN1. Also, fragments may be generated with PCRprocedures, as the sequences that are shown in SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 21. Inthis way mutation analysis may be performed.

It is known in the art that genes may vary within and among species withrespect to their nucleotide sequence. The sequence of full length HCN1cDNAs from other individuals as well as their gene sequence may now bereadily identified using the above probes and primers.

Availability of the full-length sequence now allows to provide forassays that measure whether the functional characteristics of thefull-length channel are altered. It is clear for the skilled person thatnow all kinds of functional equivalents may be genetically engineered.Therefore, the invention also comprises functional equivalents, whichare characterised in that they are capable of hybridising to at leastpart of the HCN1 sequence shown in SEQ ID NO: 1, preferably under highstringency conditions.

The present invention also includes these functional variants. Examplesof such variants are for example deletions, insertions, pointsubstitutions (single nucleotide polymorphisms) and splice variants.Also included in this term are other sequence variants, for examplesplice variants of a sequence which may differ in size through inclusionor exclusion of different exons. Identification of sequence variants andsingle nucleotide polymorphisms in particular are important for tworeasons: first by identifying the small differences that influence thetype and severity of diseases contracted by individuals; and second, bybeing able to understand the reasons behind variation of response tomedicines at the individual level. Understanding how these variations inthe genetic code influence biological systems will be the key todiscovering new medicines and enabling doctors to prescribe medicines topatients who are likely to respond.

The present invention provides such functional variants. SEQ ID NO: 3which is a full-length variant that contains a 189 bp insertion atposition 2114 in SEQ ID NO 1, indicative of a splicing variant comparedto SEQ ID NO: 1. This insertion encodes a further 63 amino acids in SEQID NO 4 in a region predicted to encode a portion of thecarboxy-terminal domain of the polypeptide (FIGS. 1 and 2). Two singlenucleotide variants are also provided in SEQ ID NO 5. The first is a C(SEQ ID NO 1) to A (SEQ ID NO 5) transition at position 107 resulting ina change of amino acid from Pro (SEQ lD NO 2) to Thr (SEQ ID NO 6) atposition 28. This occurs in a region predicted to encode the aminoterminal domain of the polypeptide (FIGS. 1 and 2). The second singlenucleotide variant is a C (SEQ ID NO 1) to T (SEQ ID NO 5) transition atposition 2064 resulting in a change of amino acid from Ser (SEQ ID NO 2)to Phe (SEQ ID NO 6). This occurs in a region predicted to encode thecarboxy-terminal domain of the polypeptide immediately upstream of thesplice variant insertion site. Both single nucleotide variants maytherefore occur in association with the splicing variant giving rise toSEQ ID NO 7 encoding the polypeptide SEQ ID NO 8. In addition, afunctional variant may comprise only one of these single nucleotidedifferences in conjunction with the splicing isoforms.

In addition, the sequences of the present invention contain a number ofother differences compared to the published sequence [1]. These includea further eleven point substitutions, corresponding to 5 amino acidchanges, and divergent sequence at the 3′ end of the published sequence[1].

Two nucleic acid fragments are considered to have hybridisable sequencesif they are capable to hybridising to one another under typicalhybridisation and wash conditions, as described, for example inManiatis, et al., pages 320-328, and 382-389, or using reducedstringency wash conditions that allow at most about 25-30% basepairmismatches, for example: 2×SSC, 0.1% SDS, room temperature twice, 30minutes each, then 2×SSC, 0.1% SDS 37° C. once, 30 minutes; then 2×SSC,room temperature twice ten minutes each. Preferably, homologous nucleicacid strands contain 15-25% basepair mismatches, even more preferably5-15% basepair mismatches. These degrees of homology can be selected byusing wash conditions of appropriate stringency for identification ofclones from gene libraries or other sources of genetic material, as iswell known in the art.

Furthermore, to accommodate codon variability, the invention alsoincludes sequences coding for the same amino acid sequences as thesequences disclosed herein. Also portions of the coding sequences codingfor individual domains of the expressed protein are part of theinvention as well as allelic and species variations thereof. Sometimes,a gene expresses different isoforms in a certain tissue which includessplicing variants, that may result in an altered 5′ or 3′ mRNA or in theinclusion of an additional exon sequence. Alternatively, the messengermight have an exon less as compared to its counterpart. These sequencesas well as the proteins encoded by these sequences all are expected toperform the same or similar functions and form also part of theinvention.

The sequence information as provided herein should not be so narrowlyconstrued as to require inclusion of erroneously identified bases. Thespecific sequence disclosed herein can be used to isolate further geneswhich in turn can be subjected to further sequence analyses therebyidentifying sequencing errors.

Thus, in one aspect, the present invention provides for a DNA sequenceencoding a full length human hyperpolarised activated ion channel of theHCN 1 subtype or functional equivalents thereof.

The DNA according to the invention may be obtained from cDNA.Alternatively, the coding sequence might be obtained from genomic DNA,or prepared using DNA synthesis techniques. The polynucleotide may alsobe in the form of RNA. The polynucleotide may be in single stranded ordouble stranded form. The single strand might be the coding strand orthe non-coding (anti-sense) strand.

The present invention further relates to polynucleotides which have atleast 80%, preferably 90% and more preferably 95% and even morepreferably at least 98% identity with SEQ ID NO:1, provided that suchpolynucleotides encode polypeptides which retain essentially the samebiological function or activity as the natural, mature protein. Evenmore preferred is the full length sequence according to SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO 5 or SEQ ID NO 7

The percentage of identity between two sequences can be determined withprograms such as DNAMAN (Lynnon Biosoft, version 3.2). Using thisprogram two sequences can be aligned using the optimal alignmentalgorithm of Smith and Waterman (1981, J. Mol. Biol, 147:195-197). Afteralignment of the two sequences the percentage identity can be calculatedby dividing the number of identical nucleotides between the twosequences by the length of the aligned sequences minus the length of allgaps.

The DNA according to the invention will be very useful for in vivo or invitro expression of the novel sequence according to the invention insufficient quantities and in substantially pure form.

In another aspect of the invention, a full length human hyperpolarisedactivated ion channel of the HCN 1 subtype or functional equivalentsthereof are provided. An example of such a polypeptide is a polypeptidesequence according to SEQ ID NO: 2. SEQ ID NO: 2 is encoded by the openreading frame of SEQ ID NO: 1 and consists of 827 amino acids.

Preferably, the polypeptides according to the invention comprise theamino acid sequence as shown in SEQ ID NO: 2.

Also functional equivalents, that is polypeptides homologous to SEQ IDNO: 2 or parts thereof having variations of the sequence while stillmaintaining functional characteristics, are included in the invention.Such variants are provided in SEQ ID NO: 4 which is a polypeptideencoded by the open reading frame of SEQ ID NO: 3, SEQ ID NO: 6 which isa polypeptide encoded by the open reading frame of SEQ ID NO: 5 and SEQID NO: 8 which is a polypeptide encoded by the open reading frame of SEQID NO: 7

The variations that can occur in a sequence may be demonstrated by (an)amino acid difference(s) in the overall sequence or by deletions,substitutions, insertions, inversions or additions of (an) amino acid(s)in said sequence. Amino acid substitutions that are expected not toessentially alter biological and immunological activities, have beendescribed. Amino acid replacements between related amino acids orreplacements which have occurred frequently in evolution are, inter aliaSer/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas ofprotein sequence and structure, Nat. Biomed. Res. Found., WashingtonD.C., 1978, vol. 5, suppl. 3). Based on this information Lipman andPearson developed a method for rapid and sensitive protein comparison(Science, 1985, 227, 1435-1441) and determining the functionalsimilarity between homologous polypeptides. It will be clear that alsopolynucleotides coding for such variants are part of the invention.

The polypeptides according to the present invention include thepolypeptides comprising SEQ ID NO: 2 but also its isoforms, i.e.polypeptides with a similarity of 70%, preferably 85%, more preferably90% even more preferably 95% or even 98%, provided these isoforms retainthe same biological functions as the sequence shown in SEQ ID NO: 2,like the sequences shown in SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.Also fragments of such polypeptides still capable of conferring thesebiological effects are included. Especially portions which still bind toligands form part of the invention. Such portions may be functional perse, e.g. in solubilized form or they might be linked to otherpolypeptides, either by known biotechnological ways or by chemicalsynthesis, to obtain chimeric proteins. Such proteins might be useful astherapeutic agent in that they may substitute the gene product inindividuals with aberrant expression of the HCN1 gene. Polypeptidesobtainable from alternative splice products are also included in thisinvention.

As an example of a functional equivalent SEQ ID NO: 4 is provided. Thispolypeptide corresponds to the open reading frame of SEQ ID NO: 3 andcontains an insertion of 63 amino acids.

Further examples of functional variants are polypeptides derived fromcoding sequences with single nucleotide variations. These include SEQ IDNO: 6 and SEQ ID NO: 8 which correspond to the open reading frames ofSEQ ID NO: 5, and SEQ ID NO: 7 respectively. Both polypeptidesincorporate amino acid substitutions at position 28 (Pro/Thr) and 680(Ser/Phe). It should be noted that either amino acid substitution mayoccur alone in the HCN1 polypeptide sequence such that any HCN1polypeptide with either one or both of these substitutions are part ofthe invention.

The sequence according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 orSEQ ID NO: 7 may also be used in the preparation of vector molecules forthe expression of the encoded protein in suitable host cells. Also,fragments of these sequences may be cloned into an expression vector andexpressed in order to determine individual functional sites along thecDNA molecule. The sequences may also be modified in that they arecloned into an expression vector either with our without the 5′ and/or3′ non-coding regions. The coding region of a cDNA clone according toSEQ ID NO: 1 is shown in SEQ ID NO: 21. The coding region of a cDNAclone according to SEQ ID NO: 3 is shown in SEQ ID NO: 22. The codingregion of a cDNA clone according to SEQ ID NO: 5 is shown in SEQ ID NO:24. The coding region of a cDNA clone according to SEQ ID NO: 7 is shownin SEQ ID NO:25. The invention therefore also provides an expressionvector comprising SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ IDNO: 25 or fragments thereof.

A wide variety of host cell and cloning vehicle combinations may beusefully employed in cloning the nucleic acid sequence coding for theHCN1 gene, the cDNA coding for HCN1 or parts thereof. For example,useful cloning vehicles may include chromosomal, non-chromosomal andsynthetic DNA sequences such as various known bacterial plasmids andwider host range plasmids and vectors derived from combinations ofplasmids and phage or virus DNA.

Vehicles for use in expression of the genes or a ligand-binding domainthereof of the present invention will further comprise control sequencesoperably linked to the nucleic acid sequence coding for a ligand-bindingdomain. Such control sequences generally comprise a promoter sequenceand sequences which regulate and/or enhance expression levels. Of coursecontrol and other sequences can vary depending on the host cellselected.

Suitable expression vectors are for example bacterial or yeast plasmids,wide host range plasmids and vectors derived from combinations ofplasmid and phage or virus DNA. Vectors derived from chromosomal DNA arealso included. Furthermore an origin of replication and/or a dominantselection marker can be present in the vector according to theinvention. The vectors according to the invention are suitable fortransforming a host cell.

Recombinant expression vectors comprising the DNA of the invention aswell as cells transformed with said DNA or said expression vector alsoform part of the present invention.

Suitable host cells according to the invention are bacterial host cells,yeast and other fungi, plant or animal host such as Chinese HamsterOvary cells or monkey cells. Especially preferred cells are HEK 293.Thus, a host cell which comprises the DNA or expression vector accordingto the invention is also within the scope of the invention. Theengineered host cells can be cultured in conventional nutrient mediawhich can be modified e.g. for appropriate selection, amplification orinduction of transcription. The culture conditions such as temperature,pH, nutrients etc. are well known to those ordinary skilled in the art.

The techniques for the preparation of the DNA or the vector according tothe invention as well as the transformation or transfection of a hostcell with said DNA or vector are standard and well known in the art, seefor instance Sambrook et al., Molecular Cloning: A laboratory Manual.2^(nd) Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1989.

The proteins according to the invention can be recovered and purifiedfrom recombinant cell cultures by common biochemical purificationmethods including ammonium sulfate precipitation, extraction,chromatography such as hydrophobic interaction chromatography, cation oranion exchange chromatography or affinity chromatography and highperformance liquid chromatography. If necessary, also protein refoldingsteps can be included.

The gene products according to the present invention can be used for thein vivo or in vitro identification of novel ligands or analogs thereof.For this purpose binding studies can be performed with cells transformedwith DNA according to the invention or an expression vector comprisingDNA according to the invention, said cells expressing the HCN1 fulllength gene product according to the invention or fragments thereof.

Alternatively also the HCN1 gene products according to the invention aswell as ligand-binding domains thereof can be used in an assay for theidentification of functional ligands or analogs for the HCN1 geneproducts.

Methods to determine binding to expressed gene products as well as invitro and in vivo assays to determine biological activity of geneproducts are well known. In general, expressed gene product is contactedwith the compound to be tested and binding, stimulation or inhibition ofa functional response is measured.

As a preferred way of detecting the binding of the ligand to theexpressed protein, also signal transduction capacity may be measured.

The present invention thus provides for a quick and economic method toscreen for therapeutic agents for the prevention and/or treatment ofdiseases related to CNS disorders such as human psychiatric andneurological dysfunction, cardiovascular dysfunction of the heart, andreproductive dysfunction and/or contraception related to I_(h) functionin testes and spermatozoa. The method is especially suited to be usedfor the high throughput screening of numerous potential compounds.

Compounds which activate or inhibit the function of HCN1 gene productmay be employed in therapeutic treatments to activate or inhibit thepolypeptides of the present invention.

Also within the scope of the invention are antibodies, especiallymonoclonal antibodies raised against the polypeptide molecule accordingto the invention. Such antibodies can be used therapeutically to inhibitHCN1 gene product function and diagnostically to detect HCN1 geneproducts.

The invention furthermore relates to the use of the HCN1 gene productsas part of a diagnostic assay for detecting clinical abnormalities orsusceptibility to any of the above disorders or investigation ofdifferent clinical outcomes in response to medical treatment related tomutations in the nucleic acid sequences encoding the HCN1 gene. Twoexamples single nucleotide variants are provided in this invention. Suchvariants or mutations may e.g. be detected by using PCR (Saiki et al.,1986, Nature, 324, 163-166). Also the relative levels of RNA can bedetermined using e.g. hybridization or quantitative PCR technology. Thepresence and the levels of the HCN1 gene products themselves can beassayed by immunological technologies such as radioimmuno assays,Western blots and ELISA using specific antibodies raised against thegene products. Such techniques for measuring RNA and protein levels arewell known to the skilled artisan.

The determination of expression levels of the HCN1 gene products andvariants thereof in individual patients may lead to fine tuning oftreatment protocols.

Also, transgenic animals may be prepared in which the expression of theHCN1 gene is altered or abolished.

EXAMPLE 1

Full-length Sequence Identification

Proprietary databases were screened by BLAST2 for the presence ofrelated human cDNA sequences using parts of the DNA sequence of thehuman HCN1 channel (Accession No AF064876) [29] and mouse HCN1(Accession number AJ225123). A single cDNA clone (SEQ ID NO: 9) wasidentified from a brain cDNA library, obtained and sequenced using anABI Prism 310 Genetic analyser (PE Biosystems). Sequencing reactionswere performed using ABI Prism BigDye Terminator cycle sequencing Readyreaction kit (PE Biosystems). Each sequencing reaction contained 300 ngcDNA clone, 3.2 pmol sequencing primer, and PE Biosystems TerminatorReady reaction mix in a final volume of 20 ul. Reactions were cycled asfollows: 25 cycles of 96° C. for 10 sec, 50° C. for 5 sec and 60° C. for4 min in a PE Biosystems GeneAmp PCR system 9700. Following cycling, theextension products were precipitated by adding 2 ul 3M NaOAc (pH 4.6)and 50 ul 95% ethanol. Products were precipitated at RT for 15 min andcollected by centrifugation at 14000 rpm for 20 min. Pellets were washed2× with 70% ethanol prior to resuspension in 20 ul

The published human HCN1 sequence and the 5′ sequence obtained byRACE-PCR was used to design primers to PCR the region of human HCN1flanked by the RACE-PCR product (SEQ ID NO: 13) and SEQ ID NO: 9 andcovered by the published sequence. Each PCR reaction contained 1× PCRbuffer (Expand High Fidelity buffer), 1.5 mM MgCl₂, 2□1 whole brainMarathon-Ready cDNA(Clontech), 400 nM primer 1 (HCN1PCRA5′-TGCTGCAGCCCGGGGTCAACAAAT-3′ (SEQ ID NO:37)), 400 nM primer 2(HCN1PCRB 5′-GAGGCGGTGGGGGAGGCATAGTGG-3′ (SEQ ID NO:38)), 400□M dATP,400 μM dCTP, 400 μM dGTP, 400□M dTTP, 5% DMSO and 2.625 units ExpandHigh Fidelity PCR enzyme mix in a total volume of 50 μl. Reactions werecycled in a MJ Research PTC-200 Thermal Cycler using the followingconditions: 94° C., 2 min and 35 cycles of 24° C. for 30 sec, 60° C. for30 sec, 72° C. for 90 sec, followed by an extension of 72° C. for 5 min.A PCR product of approximately 1.9 kb was identified, purified andsequenced as previously described (SEQ ID NO: 19). Some independentlyderived clones were also found to contain a single nucleotide variation(a C to T transition) indicated in SEQ ID NO: 20.

The full length sequence of HCN1 indicates that the cDNA consists of2484 bp open reading frame (SEQ ID NO: 22) encoding an 827 amino acidchannel. The functional equivalent sequence shown in SEQ ID NO: 3comprises an 2673 bp open reading frame (SEQ ID NO: 23) encoding a 890amino acid channel. Two single nucleotide variants were found whichaltered the amino acid sequence, and these are indicated in conjunctionwith the splice variant in SEQ ID NO: 24 and SEQ ID NO: 25. To date,four members of the hyperpolarisation-activated cation channel have beenidentified and cloned from mouse (HCN1-4). Full length clones of humanHCN-2 and HCN-4 (Accession NOs AF065164 and AJ238850) have also beenobtained whereas only a single partial sequence exists for human HCN-1.These channels share close overall homology. Analysis of the channelsequence have shown that these channels have the classic K⁺ channelstructure of 6 transmembrane domains and an ion conducting pore loop.Additionally, they possess a cyclic nucleotide binding domain. Withinthe transmembrane, pore and CNBD regions there is very high conservationamong the channel subtypes. Alignment of the HCN1 sequences according tothe invention with the other members of the HCN channel family suggestthat this sequence does encode an hyperpolarisation activated ionchannel. Regions of conservation between the human HCN channels aredenoted by asterixes in FIG. 1. The transmembrane domains, pore regionsand CNBD are underlined. Alignment of HCN1 polypeptides shows that thenovel sequences do encode HCN1 channels (FIG. 2). The single amino acidvariants are indicated in bold and are underscored with a # in thealignment in FIG. 2.

The cloned full length mouse HCN1 channel contains a polyglutaminerepeat in the C-terminal region [29]. Expansions of polyglutaminerepeats have been implicated in several neurodegenerative diseases [27].Although no polyglutamine repeat appears to be present in the novelsequence the possibility arises of allelic variation and expansion of apolyglutamine tract resulting in increased propensity toneurodegenerative diseases. The 189 bp deletion identified in the newsplice variant spans the corresponding region in the mouse HCN1 channelcontaining the polyglutamine repeat.

EXAMPLE 2

Tissue Distribution of SEQ ID NO: 1 and SEQ ID NO: 3

In order to analyze the expression of the ion channels comprising SEQ IDNO:1 and SEQ ID NO: 3 in different human tissues, Northern blot analysiswas performed. A human Multiple Tissue Expression (MTE) Array wasobtained from Clontech, which contains poly A⁺ RNA from a variety ofhuman tissues including nervous system, cardiovascular, digestive, andimmune tissue. Prehybridization was performed in Clontech's ExpressHybsupplemented with 100 μg/ml denatured sheared salmon DNA for 2 hr at 65°C. For RNA detection, ³²P labeled DNA fragments were generated with anOligolabeling kit (High Prime, Boehringer Mannheim) using a 627 bpfragment generated from SEQ ID NO: 9. This fragment starts at a Smalsite 717 bp from the 5′ end of the coding sequence and stops at the Notlcloning site (SEQ ID NO:21) and is common to both SEQ ID NO: 1 and SEQID NO: 3. The ³²P labeled probes were hybridized to the array for 16hours at 65° C. Subsequently, the array was washed with 2×SSC/1% SDS at65° C. (4×20 min) followed by 2×20 min washes at 50° C. in 0.1×SSC/0.5%SDS. Filters were exposed to a phosphorscreen for 20 hr and analysedusing Molecular Dynamics Storm Scan phosphorimage package.

From the results it can be concluded that human SEQ ID NO: 1 and SEQ IDNO: 3 are expressed predominantly in nervous tissue. Positive signalswere detected in a variety of brain regions including cerebral cortex,cerebellum, occipital lobe, temporal lobe and nucleus accumbens, Weakerexpression was observed in fetal brain. Peripherally, only weakexpression was detected in atrial tissue of the heart.

EXAMPLE 3

Tissue Distribution of SEQ ID: 1 and 3 in Nervous Tissue

In order to further analyze the expression of the ion channelscomprising SEQ ID NO:1 and SEQ ID NO: 3 in nervous tissue, Northern blotanalysis was performed. A human Multiple Tissue Northern was obtainedfrom Clontech (Human Brain MTN Blot II), which contains poly A⁺ RNA froma variety of brain regions. Prehybridization was performed in Clontech'sExpressHyb for 2 hr at 65° C. For RNA detection, ³²P labeled DNAfragments were generated as described previously for MTE array. The ³²Plabeled probes were hybridized to the filters for 16 hours at 65° C.Subsequently, the filters were washed with 2×SSC/0.05% SDS at roomtemperature (2×40 min) followed by 2×20 min washes at 50° C. in0.1×SSC/0.1% SDS. Filters were exposed to a phosphorimager screen for 20hr and analysed using Molecular Dynamics Storm Scan phosphorimagepackage.

From the results it can be concluded that human SEQ ID NO: 1 and SEQ IDNO: 3 are expressed in a variety of nervous tissue. Multiple transcriptswere detected in a cerebral cortex, cerebellum, occipital lobe, temporallobe and frontal lobe. Weaker expression was observed in putamen andmedulla, with no expression in spinal cord.

EXAMPLE 4

Functional Assay for Detecting HCN1 Channel Currents

Human HCN1 channel currents were detected under voltage-clamp using thewhole-cell configuration of the patch-clamp technique. Appropriate cells(HEK293) expressing human HCN1 channel subunits were placed in arecording chamber containing extracellular solution (Sodium chloride 135mM; Potassium chloride 5 mM; Calcium chloride 1.8 mM; Magnesium chloride0.5 mM; HEPES 5 mM) at a temperature of 20-25 degrees centigrade. Insome experiments the extracellular solution contained 30 mM Potassiumchloride, in which cases the Sodium chloride concentration was reducedto 110 mM. The glass pipettes used for recording contained pipettesolution (Sodium chloride 10 mM; Potassium chloride 130 mM; Magnesiumchloride 0.5 mM; HEPES 5 mM; EGTA 1 mM) Using standard equipment andmethodology, a cell was voltage-clamped at a holding potential of −40 mV(not allowing for liquid junction potentials). The membrane current wasrecorded throughout the experiment. To determine the current-voltagerelationship of human HCN1 channels, every 8 seconds the membranepotential was stepped sequentially to the following values (in mV) for 2or 3 seconds each: −20, −40, −60, −80, −100, −120, −140 (FIGS. 5 and 6).To determine the effects of caesium chloride (5 mM) or ZD 7288 (100micromolar) on human HCN1 channel currents, t every 8 or 10 seconds thecell potential was hyperpolarised to a potential of −120 mV (notallowing for liquid junction potentials) for 1-5 seconds and thepotential then returned to the holding potential of −40 mV. The membranecurrent was measured just after (1-10 milliseconds) the start of thehyperpolarising step and just before (10-100 milliseconds) its end. Thedifference in these two values of membrane current was taken as theamplitude of the current due to the human HCN1 channel subunits. Foreach cell, the current amplitude was determined first in normalextracellular solution and then in extracellular solution containingcaesium chloride (FIG. 3) or ZD 7288 (FIGS. 4 and 7). Alternatively, theeffect of caesium chloride (5 mM) on human HCN1 channel currents wasdetermined by measuring the current-voltage relationship in normalextracellular solution and also in extracellular solution containingcaesium chloride (FIG. 6). Human HCN1 channel currents were reduced bycaesium chloride (5 mM) and by ZD 7288 (100 micromolar) [17, 25].

A clone which has the following characteristics is considered to be anI_(h) ion channel:

Template suppression reagent (PE Biosystems) for sequencing. This cloneencoded the 3′ end of human HCN-1. The sequence is shown in SEQ ID NOs:9.

The presence of a second splice variant was confirmed by PCR usingprimers designed against the published sequence flanking either side ofthe 189 bp deletion identified in SEQ ID NO: 9 compared to the publishedsequence. Each PCR reaction contained 1×PCR buffer (Expand High Fidelitybuffer), 1.5 mM MgCl₂, 2□1 whole brain Marathon-Ready cDNA (Clontech),400 nM primer 1 and 2 (HCN1SPLICEA 5′-TGCTGCAGCCCGGGGTCAACAAAT-3′ (SEQID NO:26) and HCN1SPLICE B 5′-CTCCTGCCCCCTGCCTGAAG-3′ (SEQ ID NO:27), orHCN1SPLICEC 5′-TCTACTACGACCCCGACCTC-3′ (SEQ ID NO:28) and HCN1SPLICED5′-TGGCTCCCGACGACATCT-3′ (SEQ ID NO:29)), 400□M dATP, 400 μM dCTP, 400□MdGTP, 400□M dTTP, 5% DMSO and 2.625 units Expand High Fidelity PCRenzyme mix in a total volume of 50□1. Reactions were cycled in a MJResearch PTC-200 Thermal Cycler using the following conditions: 94° C.,2 mm and 35 cycles of 24° C. for 30 sec, 60° C. for 30 sec, 72° C. for90 sec, followed by an extension of 72° C. for 5 mm. PCR products ofapproximately 560 bp (HCN1SPLICEA and HCN1SPLICEB) and 600 bp(HCN1SPLICEC and HCN1SPLICED) were identified, purified and sequenced aspreviously described (SEQ ID NO: 17). This sequence had no deletioncompared to the published sequence. Several independently derived cloneswere observed to contain a single nucleotide variant (a C to Ttransition) shown in SEQ ID NO: 11 which results in a change in aminoacid (Ser to Phe) in SEQ ID NO: 12.

Primers were designed using the known human HCN1 sequence to attempt toobtain the 5′ end of HCN1 by RACE-PCR using Clontech SMART RACE cDNAamplification kit. SMART 5′-RACE-ready cDNA was synthesised as follows,1 μg of hippocampal polyA⁺ RNA was mixed with 2 μM 5′-RACE cDNAsynthesis primer (Clontech 5′-(T)₂₅N⁻¹N-3′) and 2 M SMART IIoligonucleotide (Clontech 5′-AAGCAGTGGTAACAACGCAGAGTACGCGGG-3′ (SEQ IDNO: 30)) in a total volume of 5 μl. Tubes were incubated at 70° C. for 2min and cooled on ice for a further 2 min. The following was then added,1× First strand buffer (50 mM Tris-HCl pH8.3, 75 mM KCl and 6 mM MgCl₂),2 mM DTT, 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM dTTP and 200 u MMLVreverse transcriptase. The reactions were incubated at 42° C. for 1.5 hrin an air incubator. The reaction product was diluted with 250 ulTricine-EDTA buffer and heated at 72° C. for 7 min prior to storage at20° C.

Each RACE-PCR reaction contained 1×PCR buffer (Clontech Advantage-GC 2PCR buffer, 40 mM Tricine-KOH pH9.2, 15 mM KOAc, 3.5 mM Mg(OAc)₂, 5%DMSO, 187.5 ng BSA, 0.005% Nonidet P-40 and 0.005% Tween-20), 200 uMdATP, 200 uM dCTP, 200 uM dGTP, 200 uM dTTP, 2.5 ul RACE-ready cDNA,Clontech Universal primer mix (20 nM long primer5′-CTAATACGACTCACTATAGGGCAAGCAGTGGTAACAACGCAGAGT-3′ (SEQ ID NO:31), 100nM short primer 5′-AAGCAGTGGTAACAACGCAGACT-3′SEQ ID NO:32), 200 nM GeneSpecific Primer (BCNG1R2, 5′-CTGGCTGTCTFfGTAAACTFITCAGATCCATFF-3′ (SEQID NO: 33), BCNG1R3 5′-CTGTAAGGGTGGATAATCCAGAAGCCTGC-3′ (SEQ ID NO: 34),BCNG R4B, 5′-TTCTGGCTCCCAAACATGCGGAGG-3′ (SEQ ID NO: 35)), 1×Advantage-GC 2 polymerase mix (1% glycerol, 0.3 mM Tris-HCl pH 8.0, 1.5mM KCl, 1 uM EDTA) and 0.5M GC-melt (Clontech) in a total volume of 50ul. Reactions were cycled in a MJ Research PTC-200 Thermal Cycler usingthe following conditions: 95° C., 3 min and 40 cycles of 94° C. for 20sec, 65° C. for 20 sec, 72° C. for 90 sec, followed by an extension of72° C. for 5 min. A PCR product of approximately 300 bp was identified,purified and sequenced as previously described. RACE-PCR generated novelsequence information of 153 bp upstream of published human HCN1 sequenceSEQ ID NO: 13.

SEQ ID NO: 13 was used to screen by BLAST2 human public EST and genomicdatabases available from EMBL [31]. A human genomic bacterial artificialchromosome (BAC) clone (RP11 -398G9: accession number AC013384) thatcontained sequence similarity was identified from the high throughputgenomic division of the EMBL release 62. The region corresponding tohuman HCN-1 was sequenced as described previously. This clone appearedto encode the 5′ end of human HCN-1 including the translation initiationcodon. The sequences are shown in SEQ ID NO: 14. Several independentlyderived clones were found to have a single nucleotide variant (a C to Atransition) indicated in SEQ ID NO: 15 resulting in a change of aminoacid from Pro to Thr.

-   1. It opens through hyperpolarisation and closes at positive voltage    values (V_(m)-10 mV;-   2. Whose activation and deactivation proceeds with a relatively slow    time course;-   3. Which conducts not only K⁺ ions but also Na⁺ ions;-   4. Which are blocked preferentially by extracellular caesium ions    rather than by extracellular barium ions.-   5. Which are directly modulated by cyclic nucleotides, particularly    cAMP and cGMP

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: CLUSTAL W Multiple Sequence Alignments of SEQ ID NO: 2 SEQ IDNO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 with human HCN1 (SEQ ID NO:36),human HCN2 (SEQ ID NO:39) and human HCN4 (SEQ ID NO: 40).

FIG. 2: CLUSTAL W Multiple Sequence Alignments of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 with rat (SEQ ID NO: 41), mouse(SEQ ID NO: 42) and partial human HCN1 (SEQ ID NO: 43) channels.

FIG. 3: Effect of caesium chloride (5 mM) on hHCN1 (full-length)currents. Whole-cell patch-clamp recording from a HEK 293F celltransfected with an expression vector containing SEQ ID NO 7. Panel A:each data point represents a measurement from one data sweep. Caesiumchloride was added to the bathing medium for the time represented by thebar above the data points. Panel B: representative data sweeps(corresponding letters in panels A & B identify the times at which thesweeps in panel B were taken). Inward membrane currents in panel B areplotted as negative quantities (following convention), but the amplitudeof hHCN1 current in panel A is plotted as a positive value.

FIG. 4: Effect of ZD 7288 (100 micromolar) on hHCN1 (full-length)currents. Whole-cell patch-clamp recording from a HEK 293F celltransfected with an expression vector containing SEQ ID NO 7. Panel A:each data point represents a measurement from one data sweep. ZD 7288was added to the bathing medium for the time represented by the barabove the data points. Panel B: representative data sweeps(corresponding letters in panels A & B identify the times at which thesweeps in panel B were taken). Inward membrane currents in panel B areplotted as negative quantities (following convention), but the amplitudeof hHCN1 current in panel A is plotted as a positive value.

FIG. 5: Current-voltage relationships of whole-cell patch-clamprecordings from HEK 293F cells transfected with DNA for hHCN1. Panel A:SEQ ID NO 5. Panel B: SEQ ID NO 7. For each panel, the upper tracesdisplay the currents evoked by the voltage steps shown in the lowertraces.

FIG. 6: Current-voltage relationship and block by caesium chloride ofhHCN1 current. Whole-cell patch-clamp recording from a HEK 293F celltransfected with DNA expressing SEQ ID NO:3. Each panel shows thecurrents evoked by voltage commands (voltage traces not shown). Panel A:normal extracellular solution. Panel B: extracellular solutioncontaining 5 mM caesium chloride. Panel C: return to normalextracellular solution. All panels from the same cell.

FIG. 7: Effect of ZD 7288 (100 micromolar) on hHCN1 current. Whole-cellpatch-clamp recording from a HEK 293 cell transfected with KDNAexpressing SEQ ID NO:3. Panel A: each data point represents ameasurement from one data sweep. ZD 7288 was added to the bathing mediumfor the time represented by the bar above the data points. Panel B:representative data sweeps (corresponding letters in panels A & Bidentify the times at which the sweeps in panel B were taken). Note thatinward membrane currents in panel B are plotted as negative quantities(following convention), but that the amplitude of hHCN1 current in panelA is plotted as a positive value.

REFERENCES

-   [1] North, R. A. (ed) (1995) Ligand- and Voltage-gated Ion Channels,    CRC Press,-   [2] Attwell, D. and Wilson, M. (1980) Journal of Physiology 309,    287-315.-   [3] Bader, C. R. and Bertrand, D. (1984) Journal of Physiology 347,    611-631.-   [4] Bal, T. and McCormick, D. A. (1996) Neuron 17, 297-308.-   [5] Bal, T. and McCormick, D. A. (1997) Journal of Neurophysiology    77, 3145-3156.-   [6] Barnes, S. and Hille, B. (1989) Journal of General Physiology    94, 719-743.-   [7] Beaumont, V. and Zucker, R.S. (2000) Nature Neuroscience 3,    133-141.-   [8] Brown, H F and Ho, W K. (1996) in: Molecular physiology and    pharmacology of cardiac ion channels and transporters. (Morad, M.,    Ebashi, S., Trautwein, W., and Kurachi, Y., Eds.) pp. 17-30 Kluwert    Academic Publishers, Dordrecht-   [9] Clapham, D. E. (1998) Neuron 21, 5-7.-   [10] DiFrancesco, D. (1981) Journal of Physiology 314, 359-376.-   [11] DiFrancesco, D. (1993) Annual Review of Physiology 55, 455-472.-   [12] DiFrancesco, D. (1996) in: Molecular physiology and    pharmacology of cardiac ion channels and transporters. (Morad, M.,    Ebashi, S., Trautwein, W., and Kurachi, Y., Eds.) pp. 31-37 Kluwer    Academic Publishers, Dordrecht-   [13] DiFrancesco, D. and Mangoni, M. (994) Journal of Physiology    474, 473-482.-   [14] Gasparini, S. and DiFrancesco, D. (1997) Pflugers    Archiv—European Journal of Physiology 435, 99-106.-   [15] Gauss, R., Seifert, R., and Kaupp, U. B. (1998) Nature 393,    583-587.-   [16] Halliwell, J. V. and Adams, P. R. (1982) Brain Research 250,    71-92.-   [17] Harris, N. C. and Constanti, A. (1995) Journal of    Neurophysiology 74, 2366-2378.-   [18] Hille, B. (1992) Ionic Channels of Excitable Membranes, Sinauer    Associates,-   [19] Ludwig, A., Zong, X., Jeglitsch, M., Hofmann, F., and    Biel, M. (1998) Nature 393, 587-591.-   [20] Luthi, A. and McCormick, D. A. (1998) Neuron 21, 9-12.-   [21] Maccaferri, G. and McBain, C. J. (1996) Journal of Physiology    497, 119-130.-   [22] Magee, J. C. (1999) Nature Neuroscience 2, 508-514.-   [23] McCormick, D. A. and Pape, H. C. (1990) Journal of Physiology    431, 291-318.-   [24] Moosmang, S., Biel, M., Hofmann, F., and Ludwig, A. (1999)    Biol. 380, 975-980.-   [25] Pape, H. C. (1996) Annual Review of Physiology 58, 299-327.-   [26] Rekling, J. C. and Feldman, J. L. (1998) Annual Review of    Physiology 60, 385-405.-   [27] Ross, C. A., Wood, J. D., Schilling, G., Peters, M. F.,    Nucifora, F. C. J., Cooper, J. K. et al. (1999) Philosophical    Transactions of the Royal Society of London—Series B: Biological    Sciences 354, 1005-1011.-   [28] Santoro, B., Grant, S. G. N., Bartsch, D., and    Kandel, E. R. (1997) Proc. 94, 14815-14820.-   [29] Santoro, B., Liu, D. T., Yao, H., Bartsch, D., Kandel, E. R.,    Siegelbaum, S. A. et al. (1998) Cell 93, 717-729.-   [30] Seifert, R., Scholten, A., Gauss, R., Mincheva, A., Lichter,    P., and Kaupp, U. B. (1999) Proc. 96, 9391-9396.-   [31] Stoesser, G., Tuli, M. A., Lopez, R., and Sterk, P. (1999)    Nucleic Acids Research 27, 18-24.-   [32] Strata, F., Atzori, M., Molnar, M., Ugolini, G., Tempia, F.,    and Cherubini, E. (1997) Journal of Neuroscience 17, 1435-1446.-   [33] Wickman, K., Nemec, J., Gendler, S. J., and    Clapham, D. E. (1998) Neuron 20, 103-114.

1. An isolated DNA sequence encoding a full length human hyperpolarizedactivated ion channel of the HCN 1 subtype, comprising: SEQ ID:
 5. 2. Anisolated DNA sequence comprising SEQ ID NO: 22 or SEQ ID NO:
 24. 3. Anisolated DNA sequence comprising SEQ ID NO: 23 or SEQ ID NO:
 25. 4. Anisolated DNA sequence comprising SEQ ID NO: 3 or SEQ ID NO:
 7. 5. Anisolated, full length human hyperpolarized activated ion channel of theHCN1 subtype, comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.6. The isolated, full length human hyperpolarized activated ion channelof the HCN 1 subtype of claim 5, comprising: SEQ ID NO:
 6. 7. Theisolated, full length hyperpolarized activated ion channel of the HCN1subtype of claim 5, comprising SEQ ID NO:8.
 8. An in vitro screeningassay for the selection of compounds that bind the human hyperpolarisedactivated ion channel of the HCN 1 subtype, comprising: providing cellsexpressing the full length human hyperpolarized activated ion channel ofthe HCN 1 subtype according to claim 5, contacting the compounds withthe full length human hyperpolarized activated ion channel of the HCN 1subtype, and determining whether the compounds bind specifically to thefull length human hyperpolarized activated ion channel of HCN 1 subtype.9. A method to screen for compounds that influence a function of thehuman hyperpolarised activated ion channel of the HCN 1 subtype,comprising: assaying compounds with the full length human hyperpolarizedactivated ion channel of the HCN 1 subtype according to claim 5 todetermine whether the compounds activate or inhibit said ion channel.10. An isolated expression vector comprising SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 23, SEQ ID NO: 25.